Testosterone (T) administration increases skeletal muscle mass in men, causing hypertrophy of type I and II fibers, and increasing myonuclei and satellite cells, while reducing fat mass. We propose that these effects are due in part to the modulation of the conversion of pluripotent cells (PPC) residing in the skeletal muscle into myogenic and adipogenic lineages. Our hypothesis is that androgens stimulate through an androgen receptor (AR) mechanism: 1) the in vitro conversion of embryonic mesodermal PPC into the myogenic lineage, while inhibiting their adipogenic potential, and these effects can be replicated in cultures of non-embryonic PPC from mouse muscle; and 2) the conversion of non-embryonic PPC into satellite-like cells, which would donate their nuclei into pre-existing fibers, both in vitro and in vivo. Our Aim 1 is to determine in vitro whether: a) T and DHT modulate embryonic non-muscle PPC differentiation into myogenic or adipogenic lineages through an AR-dependent mechanism; and b) these effects can be replicated in primary cultures of PPC from mouse muscle. For a) we will characterize in the mouse C3H/10T1/2 cell line, the effects of increasing doses of T and DHT, without or with an AR antagonist, on: i) myogenesis, utilizing early, intermediate, and late markers; and ii) adipogenesis, utilizing early and late markers. Measurements will be performed by quantitative immunocytochemistry, western blot, and RT/PCR. For b) we will perform similar determinations on PPC cultures from young mouse muscle: the regenerating muscle fibroblasts (RMF) and/or the side-population (SP) cells. Our Aim 2 is to determine whether: a) androgen stimulation of myogenic conversion of the muscle PPC in vitro generates satellite-like cells that donate their nuclei to myotubes in an AR-dependent process; and b) whether this occurs in vivo in PPC explants into mouse skeletal muscle. For a) we will label the nuclei of muscle PPC, and incubate PPC with mouse C2C12 myoblasts or myotubes, to determine whether androgens stimulate MyoD expression and the fusion of the donor nuclei into myotubes, in an AR-dependent pathway. For b), the labeled PPC will be implanted in vivo into regenerating muscle, in intact and castrated nude mice treated or not with T, applying the same end-points as in vitro. These studies would provide novel insights on the mechanisms of androgen action and have implications for potential therapeutic uses of androgens for aging-related sarcopenia and other conditions associated with loss of muscle mass and function.